JP7582073B2 - Matrix-assisted laser desorption/ionization mass spectrometry apparatus and method - Google Patents

Description

The present invention relates to a matrix assisted laser desorption/ionization (MALDI) mass spectrometry device and method.

In the MALDI method, in order to analyze analytes that do not easily absorb laser light or that are easily damaged by laser light (e.g., objects made of proteins), a sample is prepared by mixing the analyte with a matrix that absorbs laser light and is more easily ionized than the analyte, and the sample is irradiated with laser light to ionize the analyte through the interaction between the analyte in the sample, the matrix, and the laser light. The MALDI method can analyze polymeric compounds with large molecular weights without causing much dissociation, and is also highly sensitive and suitable for trace analysis, so it has been widely used in fields such as life sciences in recent years.

In the MALDI method, samples are generally prepared by mixing a solution containing a matrix with the analyte, attaching the mixture to a sample plate, and drying the mixture (evaporating the solvent in the mixture). In a sample prepared in this way, the analyte is not necessarily uniformly present on the sample plate, and may be unevenly distributed at a specific position. In addition, since the amount of sample is usually sufficiently small relative to the amount of matrix, even if the matrix and the analyte are mixed in a nearly uniform state, there may be positions in the mixture where the analyte is not present. When a matrix is applied to a living organism such as a microorganism to measure an analyte present in the organism, the distribution of the analyte is determined by the biological tissue, so the analyte will be unevenly distributed. In this way, when the analyte is unevenly distributed at a specific position, in order to perform an analysis with high sensitivity or accuracy, it is necessary to irradiate the position where the analyte is present in large amounts (unevenly distributed position) with a laser beam. However, since many of the analytes derived from living organisms and matrices suitable for ionizing them are nearly colorless and transparent, it is difficult to identify such positions.

In the invention described in Patent Document 1, a substance (i.e., a fluorescent substance) that absorbs light of a specific wavelength or wavelength range (absorption wavelength range) (e.g., ultraviolet light) and emits light of a different wavelength (e.g., visible light) is used as a matrix, and light of a wavelength range (illumination wavelength range) that includes the absorption wavelength range is irradiated onto an area that covers the entire dried sample. The light emitted from the matrix is then detected by a camera or the like to detect the uneven distribution position of the matrix (and thus the analyte).

JP 2018-036100 A

The invention described in Patent Document 1 is based on the premise that the matrix and the analyte are similarly unevenly distributed. However, in reality, this is not always the case. For example, when the mixed liquid is dried, the analyte and the matrix may separate, causing them to be unevenly distributed separately. In such cases, the invention described in Patent Document 1 cannot perform highly sensitive and highly accurate measurements of the analyte.

The problem that the present invention aims to solve is to provide a MALDI mass spectrometer and method that can perform highly sensitive and accurate analysis of an analyte in a sample in which the analyte is mixed with a matrix.

The matrix-assisted laser desorption/ionization mass spectrometry method according to the present invention, which has been made to solve the above problems, comprises the steps of:
a fluorescent staining step of fluorescently staining the analyte with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range;
a matrix preparation step of preparing a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a second predetermined absorption wavelength range;
a sample preparation step of preparing a sample on a sample plate by mixing the analyte fluorescently dyed in the fluorescent staining step with the matrix;
an illumination light irradiation step of irradiating the sample with illumination light having an illumination wavelength range including at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range;
a fluorescence intensity distribution measuring step of measuring an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying step of specifying an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
and an ionization step of irradiating the irradiation target position with the laser beam.

A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to the present invention is an apparatus for analyzing an analyte using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range, and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a predetermined second absorption wavelength range,
a sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light having an illumination wavelength range that includes at least a portion of the first absorption wavelength range and at least a portion of the second absorption wavelength range;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying unit that specifies an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position.

Another aspect of the matrix-assisted laser desorption/ionization mass spectrometry apparatus according to the present invention is an apparatus for analyzing an analyte using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range, and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a predetermined second absorption wavelength range,
A sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light having an illumination wavelength range that includes at least a portion of the first absorption wavelength range and at least a portion of the second absorption wavelength range;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
an image display unit that displays an image of the intensity distribution of the first fluorescence and the second fluorescence measured by the fluorescence intensity distribution measurement unit;
a laser beam irradiation target position designation unit that allows a user to designate an irradiation target position to be irradiated with a laser beam that ionizes the object to be analyzed in the image displayed on the image display unit;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position.

According to the present invention, a sample obtained by mixing an analyte dyed with a fluorescent material (fluorescent paint, fluorescent dye, etc.) that emits a first fluorescence with a matrix that emits a second fluorescence is irradiated with illumination light, and the analyte and matrix in the sample emit first and second fluorescence with different wavelengths. The intensity distributions of the first and second fluorescence correspond to the concentrations of the analyte and the matrix, respectively, so that highly sensitive and highly accurate measurements can be performed by determining the position of the irradiated target based on these intensity distributions.

1 is a schematic diagram of a MALDI-TOFMS, which is one embodiment of a MALDI mass spectrometer according to the present invention. 1 is a flowchart showing one embodiment of a MALDI mass spectrometry method according to the present invention. FIG. 2 is a diagram showing an example of an image displayed on a display unit in the MALDI-TOFMS of the present embodiment. FIG. 13 is a diagram showing an example in which only the overlapping region is displayed on the display section in the MALDI-TOFMS of this embodiment. FIG. 13 is a diagram showing another example of an image displayed on the display unit in the MALDI-TOFMS of this embodiment. FIG. 13 is a diagram showing an example in which only an area corresponding to an analyte is displayed on the display section in the MALDI-TOFMS of this embodiment. FIG. 13 is a diagram showing an example in which only a matrix-corresponding region is displayed on the display section in the MALDI-TOFMS of this embodiment. FIG. 13 is a schematic diagram showing the configuration of an irradiation light source and its surroundings in a modified MALDI-TOFMS. FIG. 13 is a schematic diagram showing a control section in a MALDI-TOFMS according to another modified example and some of the components connected thereto. FIG. 13 is a diagram showing an example of an image displayed on a display unit in the MALDI-TOFMS of the modified example. FIG. 13 is a schematic diagram showing a control section in a MALDI-TOFMS according to still another modified example and some of the components connected thereto.

An embodiment of a MALDI mass spectrometry method and apparatus according to the present invention will be described using Figures 1 to 10.

(1) Configuration of the MALDI Mass Spectrometer of the Present Embodiment FIG. 1 shows a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) 1 which is one embodiment of the MALDI mass spectrometer according to the present invention.

The MALDI-TOFMS1 comprises a chamber 10 whose interior is evacuated by a vacuum pump 101, and comprises within this chamber 10 a sample stage (corresponding to the sample holder) 11, a stage drive unit 12, an extraction electrode 131, an acceleration electrode 132, a flight tube 17, and an ion detector 18. The stage drive unit 12 combined with a stage drive control unit 23 (described below) corresponds to the irradiation position movement unit. Samples are prepared in each well of a sample plate 51, which has many circular wells in a plan view. This sample plate 51 is placed (held) on the sample stage 11.

The sample stage 11 can be moved in two directions, the X-axis and the Y-axis, shown in FIG. 1, by the stage drive unit 12. The extraction electrode 131 extracts the ions generated as described below in the positive direction of the Z-axis, and the acceleration electrode 132 accelerates the ions extracted by the extraction electrode 131 in the positive direction of the Z-axis. The flight tube 17 has a flight space in the Z-axis direction inside, and separates the ions according to their mass-to-charge ratio while they fly in this flight space. The ion detector 18 is provided at a position where the ions that have passed through the flight tube 17 arrive, and detects the ions that arrive with a time lag due to differences in mass-to-charge ratio. Note that while a linear type flight tube 17 is shown in FIG. 1, a reflectron type flight tube may also be used. Furthermore, a mass analyzer other than a time-of-flight type may also be used.

The MALDI-TOFMS1 further includes, outside the chamber 10, a laser light source 14, an ultraviolet light LED (corresponding to the irradiation light source) 15, and a camera (corresponding to the fluorescence intensity distribution measurement unit) 16. A first window 141 and a second window 161 are provided in the wall of the chamber 10. The laser light source 14 is disposed at a position where the laser beam it emits passes through the first window 141, and the camera 16 is disposed at a position where the visible light (first fluorescence and second fluorescence) that has passed through the second window 161 is incident.

Between the laser light source 14 and the first window 141, a movable mirror 151 is provided that can enter and exit the optical path of the laser beam emitted by the laser light source 14. When the movable mirror 151 is disposed on the optical path of the laser beam, its back surface (a surface that does not reflect light) faces the laser light source 14, and a reflective surface provided on the opposite side of the back surface faces in an inclined direction with respect to the optical path of the laser beam. Therefore, the laser beam emitted by the laser light source 14 is incident on the first window 141 when the movable mirror 151 exits the optical path of the laser beam, and is not incident on the first window 141 when the movable mirror 151 is disposed on the optical path.

The ultraviolet LED 15 is disposed in a position where, when the movable mirror 151 is disposed on the optical path of the laser beam, the illumination light (ultraviolet light) emitted by the ultraviolet LED 15 is reflected by the movable mirror 151 and enters the first window 141. When the movable mirror 151 is removed from the optical path, the illumination light does not enter the first window 141. This illumination light has an illumination wavelength range that includes at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range. The first absorption wavelength range and the second absorption wavelength range will be described later together with an explanation of the sample (a mixture of the analyte and the matrix).

In addition, a first fixed mirror 142 is disposed within the chamber 10, which reflects the laser beam or illumination light that has passed through the first window 141 and irradiates the sample on the sample stage 11, and a second fixed mirror 162 is disposed within the chamber 10, which reflects the visible light (corresponding to the first fluorescence and the second fluorescence) emitted by the sample on the sample stage 11 and causes it to enter the second window 161.

Between the ultraviolet LED 15 and the movable mirror 151, there is provided a bandpass filter 153 that passes only light of a predetermined wavelength band including the illumination wavelength range of the illumination light emitted by the ultraviolet LED 15. In addition, between the second window 161 and the camera 16, there is provided a UV cut filter 163 that blocks ultraviolet light (including the illumination light) and passes visible light (including the first fluorescent light and the second fluorescent light).

The MALDI-TOFMS1 further has a control unit 20. The control unit 20 includes an image data receiving unit 21, a laser beam irradiation target position identifying unit 22, a stage drive control unit 23, a display control unit 24, and an analysis control unit 25. The control unit 20 is embodied by hardware such as a CPU and memory, and software that executes various controls.

A fluorescence data storage unit 26 is connected to the laser beam irradiation target position identification unit 22 and the display control unit 24. A display unit (display) 27 is connected to the display control unit 24. An input unit 28 is also connected to the control unit 20. The input unit 28 can be a keyboard, a mouse, a touch panel that constitutes the display unit 27, or the like.

The image data receiving unit 21 receives image data of the image captured by the camera 16, specifically, data on the intensity of the red (R), green (G), and blue (B) components of the light received by the camera 16 for each pixel. The laser beam irradiation target position identifying unit 22 identifies the irradiation target position of the sample on the sample stage 11 to be irradiated with the laser beam based on these image data by a method described later. The stage drive control unit 23 controls the stage drive unit 12 to move the sample stage 11 so that the irradiation target position of the sample is positioned at the position on the sample stage 11 where the laser beam emitted by the laser light source 14 is irradiated. The display control unit 24 controls the display of the image on the display unit 27 as described later based on the image data received by the image data receiving unit 21. The analysis control unit 25 controls the laser light source 14, the ultraviolet light LED 15, the movable mirror 151, the stage drive control unit 23, the ion detector 18, etc. to perform MALDI mass spectrometry. The fluorescence data storage unit 26 stores the emission wavelengths (or frequencies, wave numbers) of various fluorescent materials.

(2) Operation of the MALDI Mass Spectrometer of the Present Embodiment and the MALDI Mass Analysis Method of the Present Embodiment Hereinafter, an embodiment of the MALDI mass analysis method according to the present invention will be described while explaining the operation of the MALDI-TOFMS 1 of the present embodiment.

First, prepare a sample by mixing the analyte and matrix using the following method.

The analyte is fluorescently stained with a fluorescent material before being mixed with the matrix (Step 1: fluorescent staining process). Examples of the analyte include biopolymers such as proteins, peptides, sugar chains, and nucleic acids. When a biopolymer contained in a microorganism is used as the analyte, the microorganism itself may be fluorescently stained. Such a fluorescent material is a material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range (hereinafter, the fluorescent material that fluorescently stains the analyte is referred to as the "first fluorescent material"), and a known material can be selected according to the analyte. For example, when a biopolymer contained in a microorganism is used as the analyte, acridine orange (first absorption wavelength range: wavelength range including 260 nm, first emission wavelength: wavelength in the visible light range between blue and red (varies depending on the analyte of the chromosome)) can be used as the first fluorescent material. Furthermore, when nucleic acids are the subject of analysis, the first fluorescent material can be SMOBIO's FluoroStain DNA fluorescent dye (first absorption wavelength range: 220-550 nm, first emission wavelength: wavelength within the range of 480-640 nm).

In addition, a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength is prepared by irradiating light included in a predetermined second absorption wavelength range (Step 2: matrix preparation process). For the matrix that emits the second fluorescence, a fluorescent material that emits the second fluorescence may be used as the substance that constitutes the matrix itself, or a matrix that is not a fluorescent material that emits the second fluorescence and is fluorescently dyed with a fluorescent material that emits the second fluorescence may be used (hereinafter, the fluorescent material that emits the second fluorescence is referred to as the "second fluorescent material"). For the second fluorescent material, CHCA (alpha-Cyano-4-hydroxycinnamic Acid, second absorption wavelength range: 300-400 nm, second emission wavelength: 400-490 nm), DHB (2,5-Dihydroxybenzoic Acid, second absorption wavelength range: 300-400 nm, second emission wavelength: 400-435 nm), etc. may be used.

The fluorescent staining process and the matrix preparation process may be performed in either order, or both may be performed simultaneously.

Next, the analyte dyed in the fluorescent dyeing step and the matrix prepared in the matrix preparation step are mixed in a solvent. This mixture is then dropped into one of the wells in the sample plate 51, and the solvent is evaporated (the mixture is dried), a so-called dried-droplet method is used to prepare a sample in which the analyte and matrix are mixed (step 3, sample preparation step). As mentioned above, since the sample plate 51 has many wells, it is possible to prepare a mixture for each of the many analytes in the same manner as above, and then drop the mixture into a different well for each analyte to prepare a sample. Also, instead of the dried-droplet method, a thin film method, a sandwich method, or other method may be used to prepare a sample.

The sample plate 51 with the samples prepared in the wells is held on the sample stage 11, and the chamber 10 is evacuated by the vacuum pump 101. Then, the stage drive control unit 23 moves the sample stage 11 so that one of the wells in the sample plate 51 is positioned within the illumination range described below. Next, the movable mirror 151 is placed on the optical path of the laser beam, and the ultraviolet light LED 15 emits illumination light having an illumination wavelength range including at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range. As a result, the illumination light passes through the bandpass filter 153, is reflected by the movable mirror 151, passes through the first window 141, is reflected by the first fixed mirror 142, and reaches a partial range (the illumination range) of the sample plate 51 on the sample stage 11. As a result, the illumination light is irradiated onto the samples in the wells positioned within the illumination range (step 4, illumination light irradiation process).

The sample illuminated with illumination light in this manner emits a first fluorescence due to the fluorescent material that fluorescently dyes the analyte, and a second fluorescence due to the matrix (or the fluorescent material that fluorescently dyes the matrix). These first and second fluorescence are reflected by the second fixed mirror 162, pass through the second window 161 and UV cut filter 163, and enter the camera 16. As a result, the camera 16 acquires data on the detected intensity (intensity distribution) of the first and second fluorescence for each pixel (step 5, fluorescence intensity distribution measurement process).

Here, a part of the illumination light is reflected by the sample and reaches the UV cut filter 163, but is blocked by the UV cut filter 163 and does not reach the camera 16. In addition, the light emitted by the ultraviolet LED 15 may contain visible light close to the first emission wavelength or the second emission wavelength in addition to ultraviolet light. However, by providing the bandpass filter 153 between the ultraviolet LED 15 and the sample stage 11, such visible light is not reflected by the sample (and passes through the UV cut filter 163) and does not reach the camera 16. The action of these filters prevents unnecessary light from entering the camera 16, so that the intensities of the first fluorescence and the second fluorescence can be obtained with high accuracy. Note that the present invention also includes the case where only one of the bandpass filter 153 and the UV cut filter 163 is provided, or the case where both are omitted.

The data obtained by the camera 16 is transmitted to the image data receiving unit 21, and is further transmitted from the image data receiving unit 21 to the laser beam irradiation target position identifying unit 22 and the display control unit 24.

The laser beam irradiation target position specifying unit 22 automatically specifies the irradiation target position to be irradiated with the laser beam for ionizing the analyte from the obtained image data, i.e., the intensity distribution of the first and second fluorescence on the sample plate 51 , based on a predetermined criterion (step 6, laser beam irradiation target position specifying step). Here, the intensity distribution of the first and second fluorescence is obtained by the laser beam irradiation target position specifying unit 22 acquiring the first and second emission wavelengths from the fluorescence data storage unit 26 and extracting the light intensities of those wavelengths from the image data. The correspondence between the various fluorescent materials stored in the fluorescence data storage unit 26 and the first and second fluorescent materials may be set by an operator inputting the correspondence using the input unit 28, or may be set by the laser beam irradiation target position specifying unit 22 extracting, from the data of the fluorescent materials stored in the fluorescence data storage unit 26, those having wavelengths exhibiting colors closest to the colors indicated by the image data.

The reference for the irradiation position can be, for example, a position where the intensity of the first fluorescence is equal to or greater than a predetermined first reference intensity, and the intensity of the second fluorescence is equal to or greater than a predetermined second reference intensity. In this case, by appropriately determining these values, the position will contain more than a predetermined amount of both the analyte and the matrix, and the analyte can be sufficiently ionized by irradiation with the laser beam. Alternatively, the position may be a position where the intensity of the first fluorescence is equal to or greater than a predetermined first intensity, and the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence is within a predetermined range. In this case, by appropriately determining these values, the position will contain more than a predetermined amount of the analyte, and the matrix will be present in an appropriate amount relative to the amount of the analyte, and the analyte can be sufficiently ionized by irradiation with the laser beam.

Next, the display control unit 24 causes the display unit 27 to display an image 30 showing the intensity distribution of the first fluorescence and the second fluorescence (step 7, image display process). FIG. 3 shows an example of the image 30 displayed on the display unit 27. In this image 30, a circle 31 corresponding to a well having a circular planar shape is displayed, and within the circle 31, a region where the first fluorescence is equal to or greater than the first reference intensity (analyte corresponding region 321) is displayed in a first color (e.g., red), and a region where the second fluorescence is equal to or greater than the second reference intensity (matrix corresponding region 322) is displayed in a second color (e.g., blue). A region where the first fluorescence is equal to or greater than the first reference intensity and the second fluorescence is equal to or greater than the second reference intensity (overlap region 33) may be displayed in a color obtained by mixing the first color and the second color (e.g., purple), or in a color different from the first color and the second color. The overlap region 33 may also be displayed with a light intensity stronger than that of the overlap region 33 of the analyte corresponding region 321 and the matrix corresponding region 322. Other examples of image display are described below.

The display control unit 24 further displays a mark 34 in the image 30 indicating the irradiation target position identified by the laser beam irradiation target position identification unit 22.

The order of the laser beam irradiation target position identification process (step 6) and the image display process (step 7) may be reversed. In addition, the display control unit 24 and the display unit 27 may be omitted in the device of this embodiment, and the image display process (step 7) may be omitted in the method of this embodiment.

After the laser beam irradiation target position identification process (and image display process) is completed, the stage drive control unit 23 moves the sample stage 11 so that the irradiation target position is positioned at the position where the laser beam emitted from the laser light source 14 is irradiated (step 8, laser beam irradiation target position identification process).

Then, the movable mirror 151 is removed from the optical path of the laser beam, and the laser beam is irradiated from the laser source 14 to the irradiation position, thereby ionizing the analyte through the interaction between the analyte, the matrix, and the laser light (step 9, ionization step). Then, mass analysis is performed on the ions of the analyte (step 10, mass analysis step). The method of mass analysis is the same as that in conventional MALDI, so a detailed description is omitted.

The above operations complete the analysis of the analyte in the sample in one well. If samples have been prepared in multiple wells, the stage drive control unit causes the stage drive control unit 23 to move the sample stage 11 so that the well containing the sample to be analyzed next is positioned within the illumination range, and steps 4 to 10 are performed. This operation is repeated until the operations for all samples have been completed, completing the series of analyses.

(3) Examples of Image Display in the Image Display Step Hereinafter, examples of operations and images when displaying an image on the display unit 27 other than those described in (2) will be described with respect to the image display step.

In the above example, the area where the first fluorescence is equal to or greater than the first reference intensity and the area where the second fluorescence is equal to or greater than the first reference intensity are displayed in different colors, but in order to do so, the intensities of the first fluorescence and the second fluorescence must be extracted separately from the intensity data obtained by the camera 16. In this case, if the hues of the first fluorescence and the second fluorescence are sufficiently different (e.g., red and blue), they can be distinguished relatively easily, but if the hues of the two are close, they can be distinguished using the following method.

で表される。この数式は、強度I₁及びI₂を変数とする3つの式から成る連立方程式であるため、これら3つの式のうちの2つを用いて強度I₁及びI₂を求めることができる。あるいは、これら3つの式から最小二乗法を用いて強度I₁及びI₂を求めてもよい。 First, the intensity ratio _R1 : _G1 : _B1 of the red, green, and blue components contained in the first fluorescence, and the intensity ratio _R2 : _G2 : _B2 of the red, green, and blue components contained in the second fluorescence are obtained by a preliminary experiment or the like. At each point in the image obtained by the camera 16, the first fluorescence and the second fluorescence are considered to emit light with intensities _I1 and _I2 that depend on the concentrations of the analyte and the matrix, respectively. Therefore, the intensity _Rp of the red component, _Gp of the green component, and _Bp of the blue component of the light at each point are

This formula is a simultaneous equation consisting of three equations with intensities _I1 and _I2 as variables, so intensities _I1 and _I2 can be calculated using two of these three equations. Alternatively, intensities _I1 and _I2 may be calculated from these three equations using the least squares method.

As described above, the intensity _I1 of the first fluorescence and the intensity _I2 of the second fluorescence at each point in the image are obtained, and the image shown in FIG. 3 can be displayed based on these intensities.

The image displayed on the display unit 27 is not limited to that shown in Fig. 3. For example, as shown in Fig. 4, the display control unit 24 may perform control so as to display only the overlapping region 33 within the circle 31. Also, as shown in Fig. 5, the intensities of the first fluorescence and the second fluorescence may be displayed in a plurality of gradations. In the case of such a gradation display, among the regions where the analyte corresponding region 321 and the matrix corresponding region 322 overlap, a region 331 where the intensities of the first fluorescence and the second fluorescence are equal to or greater than a predetermined gradation may be displayed in an emphasized manner. Alternatively, the number of stages showing the intensities of the first fluorescence and the second fluorescence may be sufficiently large, and the colors may be displayed in a continuously changing manner according to the intensities.

In addition, the display control unit 24 may perform control to display only the analyte corresponding region 321 (Figure 6A) or only the matrix corresponding region 322 (Figure 6B) so that the user can easily understand the distribution of the analyte or the distribution of the matrix .

In the example of FIG. 3, a mark 34 indicating the position to be irradiated with the laser beam is shown in the image displayed on the display unit 27, but this mark 34 may be omitted.

The user may be allowed to select from these various variations of the image using the input unit 28, and the selected image may be displayed on the display unit 27.

(4) Modifications The present invention is not limited to the above-described embodiment, and various modifications are possible.

For example, instead of using an irradiation light source that emits light including at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range like the ultraviolet light LED 15 used in the MALDI-TOFMS 1 of the above embodiment, an irradiation light source 150 may be used that combines a first irradiation light source 1501 that emits light including at least a part of the first absorption wavelength range and a second irradiation light source 1502 that emits light including at least a part of the second absorption wavelength range, as shown in FIG. 7. In that case, the first irradiation light source 1501 and the second irradiation light source 1502 may be turned on simultaneously, or may be turned on with a time difference (one may be turned on, then turned off, and the other may be turned on). In particular, when the first emission wavelength and the second emission wavelength are close to each other and it is difficult to distinguish between them, it is preferable to turn on the first irradiation light source 1501 and the second irradiation light source 1502 with a time difference. In the former case, a half mirror 154 is provided on the optical path from the first irradiation light source 1501, and the light from the second irradiation light source 1502 is reflected by the half mirror 154, so that the two lights can be combined and made to enter the first window 141 (the first irradiation light source 1501 and the second irradiation light source 1502 may be switched here). In the latter case, a movable mirror similar to the movable mirror 151 can be used instead of the half mirror 154.

In the above embodiment, the irradiation position of the laser beam is moved by moving the sample stage 11 with the stage driving unit 12, but instead, the irradiation position of the laser beam may be moved by changing the position or angle of the laser light source 14 or the position or angle of the first fixed mirror 142.

In addition, the MALDI-TOFMS of the modified example shown in FIG. 8 has a control unit 201 described below instead of the control unit 20 in the MALDI-TOFMS1 of the above embodiment. The control unit 201 has a laser beam irradiation target position designation unit 221 instead of the laser beam irradiation target position identification unit 22 of the control unit 20. The fluorescence data storage unit 26 is connected only to the display control unit 24. The input unit 28 is connected to the control unit 20 as in the MALDI-TOFMS1 of the above embodiment, but is particularly used when the laser beam irradiation target position designation unit 221 designates the irradiation target position as described below. Except for the configuration of the control unit 20 and the connection of the fluorescence data storage unit 26 and the input unit 28 to the control unit 20, the configuration of the MALDI-TOFMS of this modified example is the same as the configuration of the MALDI-TOFMS1 of the above embodiment, so only the control unit 201, the fluorescence data storage unit 26, and the input unit 28 are shown in FIG. 8.

In this modified MALDI-TOFMS, the laser beam irradiation target position identifying step, which is one of the operations of the MALDI-TOFMS1 of the above embodiment, is executed after the image display step. In this laser beam irradiation target position identifying step, the display control unit 24 further displays a pointer 35 in the image 30 shown in FIG. 3 (FIG. 9), and the user operates the input unit 28 (e.g., a mouse) to move the pointer 35 within the image 30, and then performs a predetermined operation (e.g., clicking the mouse button) to specify a position on the sample, and the laser beam irradiation target position identifying unit 22 then specifies that position as the irradiation target position. In this modified example, the irradiation target position is not automatically identified (step 6) as in the MALDI-TOFMS1.

The control unit 202 of the MALDI-TOFMS of the modified example shown in FIG. 10 has a reference wavelength data acquisition unit 29 in addition to the components of the control unit 20 in the MALDI-TOFMS1 of the above embodiment. The configuration of the MALDI-TOFMS of the modified example other than the control unit 202 is the same as that of the MALDI-TOFMS1 of the above embodiment (for this reason, only the control unit 202 is shown in FIG. 10). In this modified example, before analyzing the sample to be analyzed, only the sample in which the analyte is stained with the first fluorescent material or only the matrix that emits the second fluorescence when irradiated with the irradiation light is irradiated with irradiation light from the ultraviolet light LED 15, and the first fluorescence or the second fluorescence emitted by the sample is photographed by the camera 16. Then, the reference wavelength data acquisition unit 29 measures the wavelength of the first fluorescence or the second fluorescence emitted by the sample, and stores the obtained wavelength in the fluorescence data storage unit 26 as the first emission wavelength or the second emission wavelength. The first and second emission wavelengths thus obtained are used to analyze a sample that is a mixture of the actual analyte and matrix.

Furthermore, the components of each of the embodiments and modified examples described so far may be appropriately combined and used. For example, a laser beam irradiation target position designation unit 221 may be added to the MALDI-TOFMS1 of the above embodiment (providing both the laser beam irradiation target position identification unit 22 and the laser beam irradiation target position designation unit 221), allowing the user to select whether the irradiation target position is automatically identified by the laser beam irradiation target position designation unit 22 or the user specifies the irradiation target position by the laser beam irradiation target position designation unit 221.

[Aspects]
It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

(Section 1)
The matrix-assisted laser desorption/ionization mass spectrometry method according to claim 1,
a fluorescent staining step of fluorescently staining the analyte with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range;
a matrix preparation step of preparing a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a second predetermined absorption wavelength range;
a sample preparation step of preparing a sample on a sample plate by mixing the analyte fluorescently dyed in the fluorescent staining step with the matrix;
an illumination light irradiation step of irradiating the sample with illumination light having an illumination wavelength range including at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range;
a fluorescence intensity distribution measuring step of measuring an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying step of specifying an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
and an ionization step of irradiating the irradiation target position with the laser beam.

(Section 2)
A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to the second aspect of the present invention is an apparatus for analyzing an analyte by using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range, and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a predetermined second absorption wavelength range,
A sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light having an illumination wavelength range that includes at least a portion of the first absorption wavelength range and at least a portion of the second absorption wavelength range;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying unit that specifies an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position.

(Section 4)
A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to claim 4 is an apparatus for analyzing an analyte by using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light included in a predetermined first absorption wavelength range and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light included in a predetermined second absorption wavelength range,
A sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light having an illumination wavelength range that includes at least a portion of the first absorption wavelength range and at least a portion of the second absorption wavelength range;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
an image display unit that displays an image of the intensity distribution of the first fluorescence and the second fluorescence measured by the fluorescence intensity distribution measurement unit;
a laser beam irradiation target position designation unit that allows a user to designate an irradiation target position to be irradiated with a laser beam that ionizes the object to be analyzed in the image displayed on the image display unit;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position.

According to the method according to paragraph 1 and the apparatus according to paragraphs 2 and 4, a sample obtained by mixing an analyte dyed with a fluorescent material (fluorescent paint, fluorescent dye, etc.) that emits a first fluorescence with a matrix that emits a second fluorescence is irradiated with illumination light, whereby the analyte and the matrix in the sample emit first and second fluorescence having different wavelengths. The intensity distributions of the first and second fluorescence correspond to the concentrations of the analyte and the matrix, respectively, and therefore highly sensitive and highly accurate measurements can be performed by determining the position of the irradiated target based on these intensity distributions.

The "illumination light source that irradiates illumination light having an illumination wavelength range including at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range" may be a single light source that emits light including both at least a part of the first absorption wavelength range and at least a part of the second absorption wavelength range, or may be a combination of a first light source that emits light having at least a part of the first absorption wavelength range and a second light source that emits light having at least a part of the second absorption wavelength range. In the latter case, the first light source and the second light source may emit light simultaneously or at different times.

The "matrix that emits the second fluorescence" may be a matrix made of a material that emits the second fluorescence, or a matrix that is not made of a material that emits the second fluorescence but is fluorescently dyed with a material that emits the second fluorescence.

The intensity distribution of the first fluorescence and/or the second fluorescence may be obtained as a continuous numerical value or in multiple steps (discrete numerical values). Alternatively, the intensity of the first fluorescence and/or the second fluorescence may be obtained as a binary value indicating whether the first fluorescence and/or the second fluorescence is detected/not detected.

In the method according to paragraph 1, the fluorescent staining step and the matrix preparation step may be performed in any order, or both may be performed simultaneously.

The irradiation position moving unit in the device according to paragraphs 2 and 4 may be a unit that moves the sample holder, or a unit that changes the position of the laser light source or the angle of the laser beam emitted from the laser light source, or may be a combination of these.

In the device according to paragraph 2, the laser beam irradiation target position identifying section automatically identifies the irradiation target position to be irradiated with the laser beam that ionizes the analysis target based on the intensity distribution of the first fluorescence and the second fluorescence. In contrast, in the device according to paragraph 4, an image of the intensity distribution of the first fluorescence and the second fluorescence is displayed on the image display section, and the user is prompted to specify the irradiation target position of the laser beam using the laser beam irradiation target position designating section based on the image. In either case, the irradiation target position can be determined based on the intensity distribution of the first fluorescence and the second fluorescence.

(Section 3)
The matrix-assisted laser desorption/ionization mass spectrometry apparatus according to claim 3 is the apparatus according to claim 2, further comprising an image display unit that displays an image of the intensity distribution of either or both of the first fluorescence and the second fluorescence measured by the fluorescence intensity distribution measurement unit.

According to the device of paragraph 3, the intensity distribution of either or both of the first fluorescence and the second fluorescence is displayed as an image, so that the user can easily know the concentration distribution of either or both of the analyte and the matrix in the sample.

In the device according to paragraph 3 or 4, the intensity distribution of the first fluorescence and/or the second fluorescence can be displayed by one or more differences in the hue, saturation, and brightness of the color displayed on the image display unit. Alternatively, the intensity distribution may be displayed using contour lines. The intensity distribution may be displayed by a continuous change in value, or the intensity value may be displayed in multiple steps (corresponding to the display using contour lines). Furthermore, it may be displayed as a binary value, with "0" indicating an intensity value less than a predetermined value and "1" indicating an intensity value equal to or greater than the predetermined value.

(Section 5)
A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to a fifth aspect is the apparatus according to the third or fourth aspect, wherein the image display unit has a function of displaying only an area where the first fluorescence is equal to or greater than a first reference intensity and the second fluorescence is equal to or greater than a second reference intensity.

The device according to paragraph 5 displays only areas where a predetermined amount or more of both the analyte emitting the first fluorescence and the matrix emitting the second fluorescence are present, so that the user can easily grasp the area suitable for irradiating the laser beam.

(Section 6)
A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to a sixth aspect is the apparatus according to any one of the third to fifth aspects, wherein the image display unit has a function of displaying only an area where the first fluorescence is equal to or greater than a first reference intensity, or an area where the second fluorescence is equal to or greater than a second reference intensity.

The device according to paragraph 6 allows the user to easily grasp the area where a predetermined amount or more of the analyte emitting the first fluorescence is present, or the area where a predetermined amount or more of the matrix emitting the second fluorescence is present.

(Section 7)
A matrix-assisted laser desorption/ionization mass spectrometry apparatus according to a seventh aspect of the present invention is an apparatus according to any one of the second to sixth aspects of the present invention,
The fluorescence intensity distribution measuring unit
The intensities of the red, green, and blue components of the fluorescence at each point within the measured area are calculated;
The intensity of the first fluorescence and the intensity of the second fluorescence are determined for each point based on the measured intensity values for each point and intensity ratios of the red component, the green component and the blue component in each of the first fluorescence and the second fluorescence.

According to the device according to paragraph 7, for each point in the measured area, the intensity of the fluorescence at each point can be determined by dividing it into the intensity due to the first fluorescence and the intensity due to the second fluorescence based on the intensity of each of the red, green, and blue components determined from the intensity measurement values and the intensity ratio of the red, green, and blue components in each of the first fluorescence and the second fluorescence, so that the distribution of the analyte and matrix can be reliably obtained.

The intensity ratio values of the red, green, and blue components in the first fluorescence and the second fluorescence may be obtained separately in a preliminary experiment or the like and stored in a storage unit, or may be obtained by measuring only the substance that emits the first fluorescence or only the substance that emits the second fluorescence using the device according to the present invention.

1...TOFMS
10... chamber 101... vacuum pump 11... sample stage 12... stage driving unit 131... extraction electrode 132... acceleration electrode 14... laser light source 141... first window 142... first fixed mirror 15... ultraviolet light LED (irradiation light source)
150...Irradiation light source 1501...First irradiation light source 1502...Second irradiation light source 151...Movable mirror 153...Bandpass filter 154...Half mirror 16...Camera 161...Second window 162...Second fixed mirror 163...UV cut filter 17...Flight tube 18...Ion detector 20, 201, 202...Control unit 21...Image data receiving unit 22...Laser beam irradiation target position identifying unit 221...Laser beam irradiation target position designating unit 23...Stage drive control unit
24...Display control unit
25...Analysis control unit 26...Fluorescence data storage unit 27...Display unit 28...Input unit 29...Reference wavelength data acquisition unit 30...Image 31...Circle 321...Analysis target corresponding region 322...Matrix corresponding region 33...Overlap region 331...Region where the intensities of the first fluorescence and the second fluorescence are each equal to or higher than a predetermined gradation 34...Mark indicating the position of the irradiation target 35...Pointer
51 ... Sample plate

Claims (7)

a fluorescent staining step of fluorescently staining the analysis object with a fluorescent material that emits a first fluorescent light having a first emission wavelength by irradiating the object with light having a predetermined first illumination wavelength;
a matrix preparation step of preparing a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light having a second predetermined illumination wavelength;
a sample preparation step of preparing a sample on a sample plate by mixing the analyte fluorescently dyed in the fluorescent staining step with the matrix;
an illumination light irradiation step of irradiating the sample with illumination light including the first illumination wavelength and the second illumination wavelength;
a fluorescence intensity distribution measuring step of measuring an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying step of specifying an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
an ionization step of irradiating the irradiation target position with the laser beam. An apparatus for analyzing an analyte using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light having a predetermined first illumination wavelength, and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light having a predetermined second illumination wavelength,
A sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light including the first illumination wavelength and the second illumination wavelength;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
a laser beam irradiation target position specifying unit that specifies an irradiation target position to be irradiated with a laser beam that ionizes the analyte based on intensity distributions of the first fluorescent light and the second fluorescent light;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position. The matrix-assisted laser desorption/ionization mass spectrometry apparatus according to claim 2, further comprising an image display unit that displays an image of the intensity distribution of either or both of the first fluorescence and the second fluorescence measured by the fluorescence intensity distribution measurement unit. An apparatus for analyzing an analyte using a sample prepared on a sample plate by mixing an analyte fluorescently dyed with a fluorescent material that emits a first fluorescence having a first emission wavelength when irradiated with light having a predetermined first illumination wavelength, and a matrix that emits a second fluorescence having a second emission wavelength different from the first emission wavelength when irradiated with light having a predetermined second illumination wavelength,
A sample holder for holding the sample plate;
an illumination light source that irradiates the sample with illumination light including the first illumination wavelength and the second illumination wavelength;
a fluorescence intensity distribution measurement unit that measures an intensity distribution on the sample plate of the first fluorescence and the second fluorescence emitted from a sample irradiated with the illumination light;
an image display unit that displays an image of the intensity distribution of the first fluorescence and the second fluorescence measured by the fluorescence intensity distribution measurement unit;
a laser beam irradiation target position designation unit that allows a user to designate an irradiation target position to be irradiated with a laser beam that ionizes the object to be analyzed in the image displayed on the image display unit;
a laser light source that emits the laser beam;
an irradiation position moving unit that moves an irradiation position of the laser beam emitted from the laser light source to the irradiation target position. The matrix-assisted laser desorption/ionization mass spectrometry apparatus according to claim 3 or 4, wherein the image display unit has a function of displaying only an area where the first fluorescence is equal to or greater than a first reference intensity and the second fluorescence is equal to or greater than a second reference intensity. The matrix-assisted laser desorption/ionization mass spectrometry apparatus according to any one of claims 3 to 5, wherein the image display unit has a function of displaying only the area where the first fluorescence is equal to or greater than a first reference intensity, or the area where the second fluorescence is equal to or greater than a second reference intensity. The fluorescence intensity distribution measuring unit
The intensities of the red, green, and blue components of the fluorescence at each point within the measured area are calculated;
7. The matrix-assisted laser desorption/ionization mass spectrometry apparatus according to claim 2, wherein an intensity of the first fluorescence and an intensity of the second fluorescence are determined for each of the points based on the measured intensity values for each of the points and intensity ratios of a red component, a green component, and a blue component in each of the first fluorescence and the second fluorescence.

Images